Di-, tri-, and tetra-peptides having antiangiogenic activity

ABSTRACT

Compounds having the formula (SEQ ID NO: 1), which are useful for treating conditions that arise from or are exacerbated by angiogenesis, are described. Also disclosed are pharmaceutical compositions comprising these compounds, methods of treatment using these compounds, and methods of inhibiting angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/284,227, filed Oct. 30, 2002, which claims priority to U.S.Provisional Patent Application Ser. No. 60/335,034, filed on Oct. 31,2001, which is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to methods of inhibiting angiogenesis,methods of treating cancer, and compounds having activity useful fortreating conditions which arise from or are exacerbated by angiogenesis.Also disclosed are pharmaceutical compositions comprising the compoundsand methods of treatment using the compounds.

BACKGROUND OF THE INVENTION

Angiogenesis is the fundamental process by which new blood vessels areformed and is essential to a variety of normal body activities (such asreproduction, development and wound repair). Although the process is notcompletely understood, it is believed to involve a complex interplay ofmolecules which both stimulate and inhibit the growth of endothelialcells, the primary cells of the capillary blood vessels. Under normalconditions these molecules appear to maintain the microvasculature in aquiescent state (i.e., one of no capillary growth) for prolonged periodsthat may last for weeks, or in some cases, decades. However, whennecessary, such as during wound repair, these same cells can undergorapid proliferation and turnover within as little as five days.

Although angiogenesis is a highly regulated process under normalconditions, many diseases (characterized as “angiogenic diseases”) aredriven by persistent unregulated angiogenesis. Otherwise stated,unregulated angiogenesis may either cause a particular disease directlyor exacerbate an existing pathological condition. For example, thegrowth and metastasis of solid tumors have been shown to beangiogenesis-dependent. Based on these findings, there is a continuingneed for compounds which demonstrate antiangiogenic activity due totheir potential use in the treatment of various diseases such as cancer.

Peptides having angiogenesis inhibiting properties have been describedin commonly-owned WO 01/38397, WO 01/38347, WO99/61476, and U.S. patentapplication Ser. No. 09/915,956. However, it would be desirable toprepare antiangiogenic compounds having improved profiles of activityand smaller size.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of compounds havingangiogenesis-inhibiting properties. The invention provides peptides withenhanced properties of angiogenesis inhibition. In its principleembodiment, the present invention provides a compound of formula (I) (I)Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅, (SEQ ID NO:1)or a therapeutically acceptable salt thereof, wherein

-   -   Xaa₁ is selected from the group consisting of hydrogen and        R—(CH₂)_(n)—C(O)—, wherein n is an integer from 0 to 8 and R is        selected from the group consisting of alkoxy, alkyl, amino,        aryl, carboxyl, cycloalkenyl, cycloalkyl, and heterocycle;    -   Xaa₂ is selected from the group consisting of alanyl, D-alanyl,        arginyl, asparaginyl, aspartyl, citrullyl, glutaminyl, histidyl,        alloisoleucyl, isoleucyl, D-isoleucyl, leucyl, D-leucyl,        lysyl(N-epsilon acetyl), D-lysyl(N-epsilon acetyl),        lysyl(N-epsilon-nicotinyl), N-methylisoleucyl, methionyl,        norleucyl, norvalyl, ornithyl, phenylalanyl, prolyl, D-prolyl,        homoseryl, seryl, allothreonyl, and threonyl;    -   Xaa₃ is selected from the group consisting of arginyl,        D-arginyl, citrullyl, histidyl, homoarginyl, lysyl,        lysyl(N-epsilon isopropyl), ornithyl, and 3-(3-pyridyl)alanyl;    -   provided that when Xaa₃ is arginyl or D-arginyl then Xaa₂ is        other than arginyl;    -   Xaa₄ is absent or selected from the group consisting of        N-methyl-D-alanyl, 2-aminobutyryl, 2-aminoisobutyryl,        D-glutaminyl, homoprolyl, hydroxyprolyl, leucyl, phenylalanyl,        prolyl, and D-prolyl; and    -   Xaa₅ is selected from the group consisting of hydroxyl,        D-alanylamide, azaglycylamide, glycylamide, D-lysyl(N-epsilon        acetyl)amide, —NHCH(CH₃)₂, a group represented by the formula        —NH—(CH₂)_(n)—CHR¹R², and a group represented by the formula        —NHR³, wherein n is an integer from 0 to 8; R¹ is selected from        the group consisting of hydrogen, alkyl, cycloalkenyl, and        cycloalkyl; R² is selected from the group consisting of        hydrogen, alkoxy, alkyl, aryl, cycloalkenyl, cycloalkyl,        heterocycle, and hydroxyl, with the proviso that when n is 0, R²        is other than alkoxy or hydroxyl; and R³ is selected from the        group consisting of hydrogen, cycloalkenyl, cycloalkyl, and        hydroxyl.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I), or a therapeuticallyacceptable salt thereof, in combination with a therapeuticallyacceptable carrier.

In another embodiment, the present invention provides a method ofinhibiting angiogenesis in a mammal in recognized need of such treatmentcomprising administering to the mammal a therapeutically acceptableamount of a compound of formula (I), or a therapeutically acceptablesalt thereof.

In another embodiment, the present invention provides a method oftreating cancer in a mammal in recognized need of such treatmentcomprising administering to the mammal a therapeutically acceptableamount of a compound of claim 1 or a therapeutically acceptable saltthereof.

DETAILED DESCRIPTION OF THE INVENTION

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₃ is arginyl; and Xaa₁, Xaa₂, Xaa₄, and Xaa₅ areas defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is isoleucyl; Xaa₃ is arginyl; and Xaa₁, Xaa₄,and Xaa₅ are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is D-isoleucyl; Xaa₃ is arginyl; and Xaa₁,Xaa₄, and Xaa₅ are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is selected from the group consisting ofprolyl, D-lysyl(N-epsilon acetyl), lysyl(N-epsilon acetyl), andlysyl(N-epsilon-nicotinyl); Xaa₃ is arginyl; and Xaa₁, Xaa₄, and Xaa₅are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is selected from the group consisting ofD-alanyl, citrullyl, and D-leucyl; Xaa₃ is arginyl; and Xaa₁, Xaa₄, andXaa₅ are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is selected from the group consisting ofarginyl, asparaginyl, methionyl, norleucyl, ornithyl, aspartyl,glutaminyl, homoseryl, histidyl, seryl, allothreonyl, and threonyl; Xaa₃is arginyl; and Xaa₁, Xaa₄, and Xaa₅ are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₃ is citrullyl; and Xaa₁, Xaa₂, Xaa₄, and Xaa₅are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₂ is selected from the group consisting ofarginyl and lysyl(N-epsilon acetyl); Xaa₃ is citrullyl; and Xaa₁, Xaa₄,and Xaa₅ are as defined in formula (I).

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₁ is R—(CH₂)_(n)—C(O)—, wherein n is 0 and R isalkyl wherein methyl or n-butyl are preferred alkyl groups; Xaa₂ isselected from the group consisting of D-alanyl, arginyl, asparaginyl,aspartyl, citrullyl, glutaminyl, histidyl, isoleucyl, D-isoleucyl,D-leucyl, D-lysyl(N-epsilon acetyl), lysyl(N-epsilon acetyl),lysyl(N-epsilon-nicotinyl), methionyl, norleucyl, ornithyl, prolyl,homoseryl, seryl, allothreonyl, and threonyl; Xaa₃ is selected from thegroup consisting of arginyl and citrullyl; Xaa₄ is absent or prolyl; andXaa₅ is selected from the group consisting of D-alanylamide, —NHCH₂CH₃,and —NHCH(CH₃)₂.

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₁ is R—(CH₂)_(n)—C(O)—, wherein n is 0 and R isheterocycle wherein the heterocycle is 6-methylpyridinyl; Xaa₂ isselected from the group consisting of D-alanyl, arginyl, asparaginyl,aspartyl, citrullyl, glutaminyl, histidyl, isoleucyl, D-isoleucyl,D-leucyl, D-lysyl(N-epsilon acetyl), lysyl(N-epsilon acetyl),lysyl(N-epsilon-nicotinyl), methionyl, norleucyl, ornithyl, prolyl,homoseryl, seryl, allothreonyl, and threonyl; Xaa₃ is selected from thegroup consisting of arginyl and citrullyl; Xaa₄ is absent or prolyl; andXaa₅ is selected from the group consisting of D-alanylamide, —NHCH₂CH₃,and —NHCH(CH₃)₂.

In another embodiment, the present invention provides a compound offormula (I) wherein Xaa₁ is R—(CH₂)_(n)—C(O)—, wherein n is 0 and R isalkyl wherein methyl or n-butyl are preferred alkyl groups; Xaa₂ isselected from the group consisting of D-alanyl, arginyl, asparaginyl,aspartyl, citrullyl, glutaminyl, histidyl, isoleucyl, D-isoleucyl,D-leucyl, lysyl(N-epsilon acetyl), lysyl(N-epsilon-nicotinyl),methionyl, norleucyl, ornithyl, prolyl, homoseryl, seryl, allothreonyl,and threonyl; Xaa₃ is arginyl; Xaa₄ is prolyl; and Xaa₅ is selected fromthe group consisting of D-alanylamide, —NHCH₂CH₃, and —NHCH(CH₃)₂.

Definitions

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

As used in the present specification the following terms have themeanings indicated:

The term “alkoxy,” as used herein, represents an alkyl group attached tothe parent molecular moiety through an oxygen atom.

The term “alkyl,” as used herein, represents a monovalent group derivedfrom a straight or branched chain saturated hydrocarbon by the removalof a hydrogen atom. Preferred alkyl groups for the present inventioninvention are alkyl groups having from one to six carbon atoms (C₁-C₆alkyl). Alkyl groups of one to three carbon atoms (C₁-C₃ alkyl) are morepreferred for the present invention.

The term “alkylcarbonyl,” as used herein, represents an alkyl groupattached to the parent molecular moiety through a carbonyl group.

The term “amino,” as used herein, represents —NR^(a)R^(b), wherein R^(a)and R^(b) are independently selected from the group consisting ofhydrogen, alkyl, and alkylcarbonyl.

The term “aryl,” as used herein, represents a phenyl group, or abicyclic or tricyclic fused ring system wherein one or more of the fusedrings is a phenyl group. Bicyclic fused ring systems are exemplified bya phenyl group fused to a cycloalkenyl group, as defined herein, acycloalkyl group, as defined herein, or another phenyl group. Tricyclicfused ring systems are exemplified by a bicyclic fused ring system fusedto a cycloalkenyl group, as defined herein, a cycloalkyl group, asdefined herein or another phenyl group. Representative examples of arylinclude, but are not limited to, anthracenyl, azulenyl, fluorenyl,indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The arylgroups of the present invention can be optionally substituted with one,two, three, four, or five substituents independently selected from thegroup consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

The term “carbonyl,” as used herein, represents —C(O)—.

The term “carboxyl,” as used herein, represents —CO₂H.

The term “cycloalkenyl,” as used herein, refers to a non-aromatic cyclicor bicyclic ring system having three to ten carbon atoms and one tothree rings, wherein each five-membered ring has one double bond, eachsix-membered ring has one or two double bonds, each seven- andeight-membered ring has one to three double bonds, and each nine-toten-membered ring has one to four double bonds. Examples of cycloalkenylgroups include cyclohexenyl, octahydronaphthalenyl, norbornylenyl, andthe like. The cycloalkenyl groups of the present invention can beoptionally substituted with one, two, three, four, or five substituentsindependently selected from the group consisting of alkoxy, alkyl,carboxyl, halo, and hydroxyl.

The term “cycloalkyl,” as used herein, refers to a saturated monocyclic,bicyclic, or tricyclic hydrocarbon ring system having three to twelvecarbon atoms. Examples of cycloalkyl groups include cyclopropyl,cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, and the like. Thecycloalkyl groups of the present invention can be optionally substitutedwith one, two, three, four, or five substituents independently selectedfrom the group consisting of alkoxy, alkyl, carboxyl, halo, andhydroxyl.

The term “halo,” as used herein, represents F, Cl, Br, or I.

The term “heterocycle,” as used herein, refers to a five-, six-, orseven-membered ring containing one, two, or three heteroatomsindependently selected from the group consisting of nitrogen, oxygen,and sulfur. The five-membered ring has zero to two double bonds and thesix- and seven-membered rings have zero to three double bonds. The term“heterocycle” also includes bicyclic groups in which the heterocyclering is fused to an aryl group, as defined herein. The heterocyclegroups of the present invention can be attached through a carbon atom ora nitrogen atom in the group. Examples of heterocycles include, but arenot limited to, furyl, thienyl, pyrrolyl, pyrrolidinyl, oxazolyl,thiazolyl, imidazolyl, imidazolinyl, pyrazolyl, isoxazolyl,isothiazolyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl,pyridinyl, indolyl, indolinyl, benzothienyl, and the like. Theheterocycle groups of the present invention can be optionallysubstituted with one, two, three, or four substituents independentlyselected from the group consisting of alkoxy, alkyl, carboxyl, halo, andhydroxyl.

The term “hydroxyl,” as used herein, represents —OH.

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds of the present inventionwhich are water or oil-soluble or dispersible, which are suitable fortreatment of diseases without undue toxicity, irritation, and allergicresponse; which are commensurate with a reasonable benefit/risk ratio,and which are effective for their intended use. The salts can beprepared during the final isolation and purification of the compounds orseparately by reacting an amino group with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsulfonate, digluconate, glycerophosphate,hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate, lactate, maleate,mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate, and undecanoate. Also, amino groups in thecompounds of the present invention can be quaternized with methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl,diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, andsteryl chlorides, bromides, and iodides; and benzyl and phenethylbromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric.

Unless indicated otherwise by a “D” prefix, e.g., D-Ala or NMe-D-Ile,the stereochemistry of the α-carbon of the amino acids and aminoacylresidues in peptides described in this specification and the appendedclaims is the natural or “L” configuration. The Cahn-Ingold-Prelog “R”and “S” designations are used to specify the stereochemistry of chiralcenters in certain acyl substituents at the N-terminus of the peptidesof this invention. The designation “R,S” is meant to indicate a racemicmixture of the two enantiomeric forms. This nomenclature follows thatdescribed in R. S. Cahn, et al., Angew. Chem. Int. Ed. Engl., 5, 385-415(1966).

All peptide sequences are written according to the generally acceptedconvention whereby the α-N-terminal amino acid residue is on the leftand the α-C-terminal is on the right. As used herein, the term“α-N-terminus” refers to the free α-amino group of an amino acid in apeptide, and the term “α-C-terminus” refers to the free α-carboxylicacid terminus of an amino acid in a peptide.

For the most part, the names on naturally occurring and non-naturallyoccurring aminoacyl residues used herein follow the naming conventionssuggested by the IUPAC Commission on the Nomenclature of OrganicChemistry and the IUPAC-IUB Commission on Biochemical Nomenclature asset out in “Nomenclature of α-Amino Acids (Recommendations, 1974)”Biochemistry, 14 (2), (1975). To the extent that the names andabbreviations of amino acids and aminoacyl residues employed in thisspecification and appended claims differ from those suggestions, theywill be made clear to the reader. Some abbreviations useful indescribing the invention are defined below in the following Table 1.TABLE 1 Abbreviation Definition N-Ac N-acetyl Ala alanyl AlaNH₂alanylamide Arg arginyl Arg-NHCH₂CH₃ arginylethylamide Arg-NHCH(CH₃)₂arginylisopropylamide Arg(Pmc)arginyl(N^(G)-2,2,5,7,8-pentamethylchroman- 6-sulfonyl) Fmoc-Arg(Pbf)N-Fmoc-N^(G)-(2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl)arginine Asn asparaginyl Asn(Trt) asparaginyl(trityl) Aspaspartyl Asp(O-tBu) aspartyl(O-tert-butyl) Boc tert-butoxycarbonyl Citcitrullyl Cit-NHCH₂CH₃ citrullylethylamide Fmoc9-fluorenylmethyloxycarbonyl Gln glutaminyl Gln(Trt) glutaminyl(trityl)His histidyl His(Trt) histidyl(trityl) Hser homoseryl Hser(Trt)homoseryl(trityl) Ile isoleucyl aIle alloisoleucyl Leu leucyl Lys lysylLys(Ac) lysyl(N-epsilon-acetyl) Lys(Nic) lysyl(N-epsilon-nicotinyl) Memethyl Met methionyl Nle norleucyl Nva norvalyl NMeNva N-methylnorvalylOrn ornithyl Phe phenylalanyl Pro prolyl Pro-NHCH₂CH₃ prolylethylamidePro-NHCH(CH₃)₂ prolylisopropylamide 3-Pal 3-(3-pyridyl)alanyl Ser serylSer(O-tBu) seryl(O-tert-butyl) Thr threonyl Thr(O-tBu)threonyl(O-tert-butyl) alloThr allothreonyl alloThr(O-tBu)allothreonyl(O-tert-butyl) Tyr(O-tBu) tyrosyl(O-tert-butyl)

When not found in the table above, nomenclature and abbreviations may befurther clarified by reference to the Calbiochem-Novabiochem Corp. 1999Catalog and Peptide Synthesis Handbook or the Chem-Impex International,Inc. Tools for Peptide &Solid Phase Synthesis 1998-1999 Catalogue.

Compositions

The compounds of the invention, including not limited to those specifiedin the examples, possess anti-angiogenic activity. As angiogenesisinhibitors, such compounds are useful in the treatment of both primaryand metastatic solid tumors, including carcinomas of breast, colon,rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas,liver, gallbladder and bile ducts, small intestine, urinary tract(including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma) and tumors ofthe brain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas). Such compounds may also be useful in treating solidtumors arising from hematopoietic malignancies such as leukemias (i.e.,chloromas, plasmacytomas and the plaques and tumors of mycosisfungosides and cutaneous T-cell lymphoma/leukemia) as well as in thetreatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas). Inaddition, these compounds may be useful in the prevention of metastasesfrom the tumors described above either when used alone or in combinationwith radiotherapy and/or other chemotherapeutic agents.

Further uses include the treatment and prophylaxis of autoimmunediseases such as rheumatoid, immune and degenerative arthritis; variousocular diseases such as diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, retrolental fibroplasia,neovascular glaucoma, rubeosis, retinal neovascularization due tomacular degeneration, hypoxia, angiogenesis in the eye associated withinfection or surgical intervention, and other abnormalneovascularization conditions of the eye; skin diseases such aspsoriasis; blood vessel diseases such as hemagiomas, and capillaryproliferation within atherosclerotic plaques; Osler-Webber Syndrome;myocardial angiogenesis; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; and wound granulation. Other usesinclude the treatment of diseases characterized by excessive or abnormalstimulation of endothelial cells, including not limited to intestinaladhesions, Crohn's disease, atherosclerosis, scleroderma, andhypertrophic scars, i.e., keloids. Another use is as a birth controlagent, by inhibiting ovulation and establishment of the placenta. Thecompounds of the invention are also useful in the treatment of diseasesthat have angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minutesalia quintosa) and ulcers (Helicobacter pylori).The compounds of the invention are also useful to reduce bleeding byadministration prior to surgery, especially for the treatment ofresectable tumors.

The compounds of the invention may be used in combination with othercompositions and procedures for the treatment of diseases. For example,a tumor may be treated conventionally with surgery, radiation orchemotherapy combined with a peptide of the present invention and then apeptide of the present invention may be subsequently administered to thepatient to extend the dormancy of micrometastases and to stabilize andinhibit the growth of any residual primary tumor. Additionally, thecompounds of the invention may be combined with pharmaceuticallyacceptable excipients, and optionally sustained-release matrices, suchas biodegradable polymers, to form therapeutic compositions.

A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid-base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. A sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polypeptides,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the present invention may be employedin pure form or, where such forms exist, in pharmaceutically acceptablesalt form. By a “therapeutically effective amount” of the compound ofthe invention is meant a sufficient amount of the compound to treat anangiogenic disease, (for example, to limit tumor growth or to slow orblock tumor metastasis) at a reasonable benefit/risk ratio applicable toany medical treatment. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular patient will depend upon a variety of factors includingthe disorder being treated and the severity of the disorder; activity ofthe specific compound employed; the specific composition employed, theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidential with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.

Alternatively, a compound of the present invention may be administeredas pharmaceutical compositions containing the compound of interest incombination with one or more pharmaceutically acceptable excipients. Apharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The compositions may be administeredparenterally, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),rectally, or bucally. The term “parenteral” as used herein refers tomodes of administration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

Pharmaceutical compositions for parenteral injection comprisepharmaceutically-acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity may be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorption,such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide,poly(orthoesters), poly(anhydrides), and (poly)glycols, such as PEG.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.

The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Compositions for topicaladministration, including those for inhalation, may be prepared as a drypowder which may be pressurized or non-pressurized. In non-pressurizedpowder compositions, the active ingredient in finely divided form may beused in admixture with a larger-sized pharmaceutically-acceptable inertcarrier comprising particles having a size, for example, of up to 100micrometers in diameter. Suitable inert carriers include sugars such aslactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers.

Alternatively, the composition may be pressurized and contain acompressed gas, such as nitrogen or a liquified gas propellant. Theliquified propellant medium and indeed the total composition ispreferably such that the active ingredient does not dissolve therein toany substantial extent. The pressurized composition may also contain asurface active agent, such as a liquid or solid non-ionic surface activeagent or may be a solid anionic surface active agent. It is preferred touse the solid anionic surface active agent in the form of a sodium salt.

A further form of topical administration is to the eye. A compound ofthe invention is delivered in a pharmaceutically acceptable ophthalmicvehicle, such that the compound is maintained in contact with the ocularsurface for a sufficient time period to allow the compound to penetratethe corneal and internal regions of the eye, as for example the anteriorchamber, posterior chamber, vitreous body, aqueous humor, vitreoushumor, cornea, iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour.

Compositions for rectal or vaginal administration are preferablysuppositories which may be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active compound.

Compounds of the present invention may also be administered in the formof liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multi-lamellar hydrated liquid crystals that are dispersed inan aqueous medium. Any non-toxic, physiologically-acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they may also be used in combination withone or more agents which are conventionally administered to patients fortreating angiogenic diseases. For example, the compounds of theinvention are effective over the short term to make tumors moresensitive to traditional cytotoxic therapies such as chemicals andradiation. The compounds of the invention also enhance the effectivenessof existing cytotoxic adjuvant anti-cancer therapies. The compounds ofthe invention may also be combined with other antiangiogenic agents toenhance their effectiveness, or combined with other antiangiogenicagents and administered together with other cytotoxic agents. Inparticular, when used in the treatment of solid tumors, compounds of theinvention may be administered with IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, angioinhibins, TNP-470, pentosanpolysulfate, platelet factor 4, LM-609, SU-5416, CM-101, Tecogalan,plasminogen-K-5, vasostatin, vitaxin, vasculostatin, squalamine,marimastat or other MMP inhibitors, anti-neoplastic agents such as alphainteferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, cisplatin, taxol, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine, andthe like as well as with radiation.

Total daily dose of the compositions of the invention to be administeredto a human or other mammal host in single or divided doses may be inamounts, for example, from 0.0001 to 300 mg/kg body weight daily andmore usually 1 to 300 mg/kg body weight.

It will be understood that agents which can be combined with thecompound of the present invention for the inhibition, treatment orprophylaxis of angiogenic diseases are not limited to those listedabove, include in principle any agents useful for the treatment orprophylaxis of angiogenic diseases.

Determination of Biological Activity

In Vitro Assay for Angiogenic Activity

The human microvascular endothelial cell (HMVEC) migration assay was runaccording to the procedure of S. S. Tolsma, O. V. Volpert, D. J. Good,W. F. Frazier, P. J. Polverini and N. Bouck, J. Cell Biol. 1993, 122,497-511.

The HMVEC migration assay was carried out using Human MicrovascularEndothelial Cells-Dermal (single donor) and Human MicrovascularEndothelial Cells, (neonatal). The HMVEC cells were starved overnight inDME containing 0.01% bovine serum albuminutes (BSA). Cells were thenharvested with trypsin and resuspended in DME with 0.01% BSA at aconcentration of 1.5×10⁶ cells per mL. Cells were added to the bottom ofa 48 well modified Boyden chamber (Nucleopore Corporation, Cabin John,Md.). The chamber was assembled and inverted, and cells were allowed toattach for 2 hours at 37° C. to polycarbonate chemotaxis membranes (5 μmpore size) that had been soaked in 0.01% gelatin overnight and dried.The chamber was then reinverted, and test substances (total volume of 50μL), including activators, 15 ng/mL bFGF/VEGF, were added to the wellsof the upper chamber. The apparatus was incubated for 4 hours at 37° C.Membranes were recovered, fixed and stained (Diff Quick, FisherScientific) and the number of cells that had migrated to the upperchamber per 3 high power fields counted. Background migration to DME+0.1BSA was subtracted and the data reported as the number of cells migratedper 10 high power fields (400×) or, when results from multipleexperiments were combined, as the percent inhibition of migrationcompared to a positive control.

Representative compounds of the present invention inhibited humanendothelial cell migration in the above assay by at least 40% whentested at a concentration of 1 nM. More preferred compounds inhibitedhuman endothelial cell migration by approximately 80% to 90% when testedat a concentration of 1 nM, and the most preferred compound inhibitedhuman endothelial cell migration by approximately 70% or more whentested at a concentration of 0.1 nM. As shown by these results, thecompounds of the present invention demonstate an enhanced ability toinhibit angiogenesis as compared to previously described antiangiogenicpeptides.

Synthesis of the Peptides

This invention is intended to encompass compounds having formula (I)when prepared by synthetic processes or by metabolic processes.Preparation of the compounds of the invention by metabolic processesinclude those occurring in the human or animal body (in vivo) orprocesses occurring in vitro.

The polypeptides of the present invention may be synthesized by manytechniques that are known to those skilled in the art. For solid phasepeptide synthesis, a summary of the many techniques may be found in J.M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W.H. FreemanCo. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins andPeptides, vol. 2, p. 46, Academic Press (New York), 1973. For classicalsolution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1,Academic Press (New York), 1965.

Reagents, resins, amino acids, and amino acid derivatives arecommercially available and can be purchased from Chem-ImpexInternational, Inc. (Wood Dale, Ill., U.S.A.) or Calbiochem-NovabiochemCorp. (San Diego, Calif., U.S.A.) unless otherwise noted herein.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the finalpolypeptide. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide.

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis. In this particularlypreferred method the α-amino function is protected by an acid or basesensitive group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation, while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,(α,α)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is preferred.

Particularly preferred side chain protecting groups are: for arginine:N^(G)-2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), and2,2,4,6,7-pentamethyldihydrobenzofuran-S-sulfonyl (Pbf).

In the solid phase peptide synthesis method, the C-terminal amino acidis attached to a suitable solid support or resin. Suitable solidsupports useful for the above synthesis are those materials which areinert to the reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as being insoluble in themedia used. The preferred solid support for synthesis of C-terminalcarboxyl peptides is Sieber amide resin or Sieber ethylamide resin. Thepreferred solid support for C-terminal amide peptides is Sieberethylamide resin available from Novabiochem Corporation.

The C-terminal amino acid is coupled to the resin by means of a couplingmediated by N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC),[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HATU), orO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT), N-methylmorpholine (NMM),benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), for about1 to about 24 hours at a temperature of between 10° C. and 50° C. in asolvent such as dichloromethane or DMF.

When the solid support is Sieber amide or Sieber ethylamide resin, theFmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the C-terminal amino acid as described above. Thepreferred reagents used in the coupling to the deprotected4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinare O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) with 1-hydroxybenzotriazole (HOBT, 1 equiv.), or[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HATU, 1 equiv.) with N-methylmorpholine (1 equiv.)in DMF.

The coupling of successive protected amino acids can be carried out inan automatic polypeptide synthesizer as is well known in the art. In apreferred embodiment, the α-amino function in the amino acids of thegrowing peptide chain are protected with Fmoc. The removal of the Fmocprotecting group from the N-terminal side of the growing peptide isaccomplished by treatment with a secondary amine, preferably piperidine.Each protected amino acid is then introduced in about 3-fold molarexcess and the coupling is preferably carried out in DMF. The couplingagent is normallyO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) or[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HATU, 1 equiv.) in the presence ofN-methylmorpholine (NMM, 1 equiv.).

At the end of the solid phase synthesis, the polypeptide is removed fromthe resin and deprotected, either in succession or in a singleoperation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the resin-boundpolypeptide with a cleavage reagent, for example trifluoroacetic acidcontaining thianisole, water, or ethanedithiol.

In cases where the C-terminus of the polypeptide is an alkylamide, theresin is cleaved by aminolysis with an alkylamine. Alternatively, thepeptide may be removed by transesterification, e.g. with methanol,followed by aminolysis or by direct transamidation. The protectedpeptide may be purified at this point or taken to the next stepdirectly. The removal of the side chain protecting groups isaccomplished using the cleavage cocktail described above.

The fully deprotected peptide is purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivitized polystyrene-divinylbenzene(for example, AMBERLITE® XAD); silica gel adsorption chromatography; ionexchange chromatography on carboxymethylcellulose; partitionchromatography, e.g., on SEPHADEX® G-25, LH-20 or countercurrentdistribution; high performance liquid chromatography (HPLC), especiallyreverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phasecolumn packing.

The foregoing may be better understood in light of the examples whichare meant to describe compounds and process which can be carried out inaccordance with the invention and are not intended as a limitation onthe scope of the invention in any way.

Abbreviations which have been used the following examples are: DMF forN,N-dimethylformamide; HBTU forO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; NMMfor N-methylmorpholine; and TFA for trifluoroacetic acid.

EXAMPLE 1 N-Ac-Ile-Arg-Pro-NHCH₂CH₃

In the reaction vessel of a Rainin peptide synthesizer was placedFmoc-Pro-Sieber ethylamide resin (0.25 g, 0.4 mmol/g loading). The resinwas solvated with DMF and amino acids were coupled sequentiallyaccording to the following synthetic cycle:

-   -   (1) 3×1.5 minute washes with DMF;    -   (2) 2×15 minute deprotection using 20% piperidine;    -   (3) 6×3 minute washes with DMF;    -   (4) addition of amino acid;    -   (5) activation of amino acid with 0.4 M HBTU/NMM and coupling;    -   (6) 3×1.5 minute washes with DMF.

The protected amino acids were coupled to the resin in the followingorder: Protected Amino Acid Coupling time Fmoc-Arg(Pmc) 30 minutesFmoc-Ile 30 minutes acetic acid 30 minutes

Upon completion of the synthesis the peptide was cleaved from the resinusing a mixture of (95:2.5:2.5) TFA/anisole/water for 3 hours. Thepeptide solution was concentrated under vacuum and then precipitatedwith diethyl ether and filtered. The crude peptide was purified by HPLCusing a C-18 column and a solvent mixture varying over 50 minutes in agradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-Ile-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt=0.91 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e454 (M+H)⁺; Amino Acid Anal.: 0.93 Ile; 1.03 Arg; 1.01 Pro.

EXAMPLE 2 N-(6-methylnicotinyl)-D-Ile-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting 6-methylnicotinic acidfor acetic acid and Fmoc-D-Ile for Fmoc-Ile in Example 1. After workupthe crude peptide was purified by HPLC using a C-18 column and a solventmixture varying over 50 minutes in a gradient of 5% to 100%acetonitrile/water containing 0.01% TFA. The pure fractions werelyophilized to provide N-(6-methylnicotinyl)-D-Ile-Arg-Pro-NHCH₂CH₃ asthe trifluoracetate salt: Rt=3.67 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 531.2 (M+H)⁺; Amino Acid Anal.: 0.99 Ile; 0.96 Arg; 1.05 Pro.

EXAMPLE 3 N-Ac-D-Ile-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting Fmoc-D-Ile for Fmoc-Ilein Example 1. After workup the crude peptide was purified by HPLC usinga C-18 column and a solvent mixture varying over 50 minutes in agradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-D-Ile-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt=0.94 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e454.2 (M+H)⁺; Amino Acid Anal.: 1.03 Ile; 1.01 Arg; 1.01 Pro.

EXAMPLE 4 N-(6-methylnicotinyl)-Ile-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting 6-methylnicotinic acidfor acetic acid in Example 1. After workup the crude peptide waspurified by HPLC using a C-18 column and a solvent mixture varying over50 minutes in a gradient of 5% to 100% acetonitrile/water containing0.01% TFA. The pure fractions were lyophilized to provideN-(6-methylnicotinyl)-Ile-Arg-Pro-NHCH₂CH₃ as the trifluoracetate salt:Rt=3.43 minutes (gradient varying over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA), MS (ESI) rn/e 531.2 (M+H)⁺;Amino Acid Anal.: 0.92 Ile; 0.99 Arg; 0.97 Pro.

EXAMPLE 5 N-Ac-Pro-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting Fmoc-Pro for Fmoc-Ilein Example 1. After workup the crude peptide was purified by HPLC usinga C-18 column and a solvent mixture varying over 50 minutes in agradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-Pro-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt=2.75 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA), MS (ESI) m/e438.1 (M+H)⁺.

EXAMPLE 6 N-Ac-D-Ala-Arg-Pro-NHCH₂ CH₃

The desired product was prepared by substituting Fmoc-D-Ala for Fmoc-Ilein Example 1 After workup the crude peptide was purified by HPLC using aC-18 column and a solvent mixture varying over 50 minutes in a gradientof 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-D-Ala-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt=1.99 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e412 (M+H)⁺.

EXAMPLE 7 N-Ac-D-Leu-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting Fmoc-D-Leu for Fmoc-Ilein Example 1. After workup the crude peptide was purified by HPLC usinga C-18 column and a solvent mixture varying over 50 minutes in agradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-D-Leu-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt==3.46 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e454.1 (M+H)⁺.

EXAMPLE 8 N-Ac-Lys(Ac)-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting Fmoc-Lys(Ac) forFmoc-Ile in Example 1. After workup the crude peptide was purified byHPLC using a C-18 column and a solvent mixture varying over 50 minutesin a gradient of 5% to 100% acetonitrile/water containing 0.01% TFA. Thepure fractions were lyophilized to provide N-Ac-Lys(Ac)-Arg-Pro-NHCH₂CH₃as the trifluoracetate salt: Rt=2.72 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 511.2 (M+H)⁺.

EXAMPLE 9 N-Ac-Cit-Arg-Pro-NHCH₂CH₃

The desired product was prepared by substituting Fmoc-Cit for Fmoc-Ilein Example 1. After workup the crude peptide was purified by HPLC usinga C-18 column and a solvent mixture varying over 50 minutes in agradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The purefractions were lyophilized to provide N-Ac-Cit-Arg-Pro-NHCH₂CH₃ as thetrifluoracetate salt: Rt=2.02 minutes (gradient varying over 10 minutesfrom 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e498.1 (M+H)⁺.

EXAMPLE 10 N-Ac-D-Lys(Ac)-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-D-Lys(Ac) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-D-Lys(Ac)-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=0.76minutes (gradient varying over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 511.3 (M+H)⁺;Amino Acid Anal.: 0.91 Lys; 1.01 Arg; 1.00 Pro.

EXAMPLE 11 N-Ac-Thr-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Thr(O-tBu) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Thr-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.55 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 442.2 (M+H)⁺; Amino Acid Anal.: 0.54Thr; 1.00 Arg; 0.98 Pro.

EXAMPLE 12 N-Ac-Ser-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Ser(O-tBu) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Ser-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.27 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 428.2 (M+H)⁺; Amino Acid Anal.: 0.39Ser; 1.05 Arg; 1.02 Pro.

EXAMPLE 13 N-Ac-Nle-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substituting Fmoc-Nlefor Fmoc-Ile. Upon completion of the synthesis the peptide was cleavedfrom the resin using a mixture of (95:2.5:2.5) TFA/anisole/water for 3hr. The peptide solution was concentrated in vacuo and then precipitatedwith diethyl ether. The crude peptide was purified by HPLC using C-18column and with a solvent mixture varying over 50 minutes in a gradientfrom 5% to 100% acetonitrile-water containing 0.01% TFA. The purefractions were lyophilized to give N-Ac-Nle-Arg-Pro-NHCH₂CH₃ astrifluoroacetate salt: R_(t)=2.27 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 454.2 (M+H)⁺; Amino Acid Anal.: 1.02 Nle; 1.00 Arg; 1.02 Pro.

EXAMPLE 14 N-Ac-Orn-Arg-Pro-NHCH₂CH₃

The procedure described in example 1 was used but substitutingFmoc-Orn(Boc) for Fmoc-Ile. Upon completion of the synthesis the peptidewas cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Orn-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.27 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 455.3 (M+H)⁺; Amino Acid Anal.: 0.97Orn; 1.06 Arg; 1.02 Pro.

EXAMPLE 15 N-Ac-Asp-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Asp(O-tBu) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Asp-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.27 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 456.2 (M+H)⁺; Amino Acid Anal.: 1.02Asp; 1.01 Arg; 1.01 Pro.

EXAMPLE 16 N-Ac-Gln-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Gln(Trt) for Fmoc-Ile. Upon completion of the synthesis the peptidewas cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Gln-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.24 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 456.2 (M+H)⁺; Amino Acid Anal.: 1.00Glu; 1.05 Arg; 1.03 Pro.

EXAMPLE 17 N-Ac-Lys(Nic)-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Lys(N-epsilon-nicotinyl) for Fmoc-Ile. Upon completion of thesynthesis the peptide was cleaved from the resin using a mixture of(95:2.5:2.5) TFA/anisole/water for 3 hr. The peptide solution wasconcentrated in vacuo and then precipitated with diethyl ether. Thecrude peptide was purified by HPLC using C-18 column and with a solventmixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions werelyophilized to give N-Ac-Lys(Nic)-Arg-Pro-NHCH₂CH₃ asdi-trifluoroacetate salt: R_(t)=2.95 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 574.3 (M+H)⁺; Amino Acid Anal.: 0.93 Lys; 1.02 Arg; 0.99 Pro.

EXAMPLE 18 N-Ac-Asn-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Asn(Trt) for Fmoc-Ile. Upon completion of the synthesis the peptidewas cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Asn-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.10 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 455.2 (M+H)⁺; Amino Acid Anal.: 1.02Asp; 0.98 Arg; 0.97 Pro.

EXAMPLE 19 N-Ac-Met-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substituting Fmoc-Metfor Fmoc-Ile. Upon completion of the synthesis the peptide was cleavedfrom the resin using a mixture of (95:2.5:2.5) TFA/anisole/water for 3hr. The peptide solution was concentrated in vacuo and then precipitatedwith diethyl ether. The crude peptide was purified by HPLC using C-18column and with a solvent mixture varying over 50 minutes in a gradientfrom 5% to 100% acetonitrile-water containing 0.01% TFA. The purefractions were lyophilized to give N-Ac-Met-Arg-Pro-NHCH₂CH₃ astrifluoroacetate salt: R_(t)=3.55 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 472.2 (M+H)+; Amino Acid Anal.: 0.96 Met; 1.03 Arg; 1.05 Pro.

EXAMPLE 20 N-Ac-Hser-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Hser(Trt) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-Hser-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.19 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 442.2 (M+H)⁺; Amino Acid Anal.: 0.45Hser; 1.01 Arg; 1.00 Pro.

EXAMPLE 21 N-Ac-alloThr-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-alloThr(O-tBu) for Fmoc-Ile. Upon completion of the synthesis thepeptide was cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-alloThr-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.29minutes (gradient varying over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 442.2 (M+H)⁺;Amino Acid Anal.: 0.59 Thr; 1.01 Arg; 1.01 Pro.

EXAMPLE 22 N-Ac-His-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-His(Trt) for Fmoc-Ile. Upon completion of the synthesis the peptidewas cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution was concentrated invacuo and then precipitated with diethyl ether. The crude peptide waspurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions were lyophilized to giveN-Ac-His-Arg-Pro-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=2.40 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 478.2 (M+H)⁺.

EXAMPLE 23 N-Ac-Arg-Cit-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Arg(Pmc) for Fmoc-Ile and Fmoc-Cit for Fmoc-Arg(Pmc). Uponcompletion of the synthesis the peptide was cleaved from the resin usinga mixture of (95:2.5:2.5) TFA/anisole/water for 3 hr. The peptidesolution was concentrated in vacuo and then precipitated with diethylether. The crude peptide was purified by HPLC using C-18 column and witha solvent mixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions werelyophilized to give N-Ac-Arg-Cit-Pro-NHCH₂CH₃ as trifluoroacetate salt:R_(t)=2.36 minutes (gradient varying over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 498.3 (M+H)⁺.

EXAMPLE 24 N-Ac-Ile-Arg-Pro-NHCH(CH₃)₂

The procedure described in Example 1 was used but substitutingFmoc-Pro-[4-(4-N-isopropylamino)methyl-3-methoxyphenoxy]butyryl AM resininstead of Fmoc-Pro Sieber ethylamide resin. Upon completion of thesynthesis the peptide was cleaved from the resin using a mixture of(95:2.5:2.5) TFA/anisole/water for 3 hr. The peptide solution wasconcentrated in vacuo and then precipitated with diethyl ether. Thecrude peptide was purified by HPLC using C-18 column and with a solventmixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions werelyophilized to give N-Ac-Ile-Arg-Pro-NHCH(CH₃)₂ as trifluoroacetatesalt: R_(t)=4.23 minutes (gradient varying over 10 minutes from 20% to80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 468.3 (M+H)⁺.

EXAMPLE 25 N-Valeryl-Ile-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 was used but substituting valericacid for acetic acid. Upon completion of the synthesis the peptide wascleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution was concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide was purified by HPLCusing C-18 column and with a solvent mixture varying over 50 minutes ina gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. Thepure fractions were lyophilized to give N-valeryl-Ile-Arg-Pro-NHCH₂CH₃as trifluoroacetate salt: R_(t)=4.511 minutes (gradient varying over 10minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS(ESI) m/e 496.3 (M+H)⁺.

EXAMPLE 26 N-Ac-Ile-Arg-Pro-D-AlaNH₂

The procedure described in Example 1 was used but substitutingFmoc-D-Ala-Sieber amide resin instead of Fmoc-Pro-Sieber ethylamideresin and coupling with Fmoc-Pro before coupling with Fmoc-Arg(Pmc).Upon completion of the synthesis the peptide was cleaved from the resinusing a mixture of (95:2.5:2.5) TFA/anisole/water for 3 hr. The peptidesolution was concentrated in vacuo and then precipitated with diethylether. The crude peptide was purified by HPLC using C-18 column and witha solvent mixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions werelyophilized to give N-Ac-Ile-Arg-Pro-D-AlaNH₂ as trifluoroacetate salt:R_(t)=3.56 minutes (gradient varying over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 497.3 (M+H)⁺;Amino Acid Anal.: 1.02 Ile; 1.03 Arg; 1.02 Pro; 0.99 Ala.

EXAMPLE 27 N-Ac-Lys(Ac)-Arg-NHCH₂CH₃

The procedure described in Example 1 was used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, Fmoc-Lys(Ac) for Fmoc-Ile and omittingthe coupling with Fmoc-Arg(Pmc). Upon completion of the synthesis thepeptide can be cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution can be concentrated invacuo and then precipitated with diethyl ether. The crude peptide can bepurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Lys(Ac)-Arg-NHCH₂CH₃ as trifluoroacetate salt: R_(t)=0.61 minutes(gradient varying over 10 minutes from 20% to 80% acetonitrile/watercontaining 0.01% TFA); MS (ESI) m/e 414.1 (M+H)⁺.

EXAMPLE 28 N-Ac-Ile-Arg-NHCH₂CH₃

The procedure described in Example 1 can be used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin and omitting the coupling withFmoc-Arg(Pmc). Upon completion of the synthesis the peptide can becleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution can be concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide can be purified byHPLC using C-18 column and with a solvent mixture varying over 50minutes in a gradient from 5% to 100% acetonitrile-water containing0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Ile-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 29 N-Ac-Lys(Ac)-Cit-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Cit-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, Fmoc-Lys(Ac) for Fmoc-Ile and omittingthe coupling with Fmoc-Arg(Pmc). Upon completion of the synthesis thepeptide can be cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution can be concentrated invacuo and then precipitated with diethyl ether. The crude peptide can bepurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Lys(Ac)-Cit-NHCH₂CH₃.

EXAMPLE 30 N-Ac-Lys(Nic)-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, FmocLys(N-epsilon-nicotinyl) forFmoc-Ile and omitting the coupling with Fmoc-Arg(Pmc). Upon completionof the synthesis the peptide can be cleaved from the resin using amixture of (95:2.5:2.5) TFA/anisole/water for 3 hr. The peptide solutioncan be concentrated in vacuo and then precipitated with diethyl ether.The crude peptide can be purified by HPLC using C-18 column and with asolvent mixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions can belyophilized to give N-Ac-Lys(Nic)-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 31 N-Ac-Ile-Arg-NHCH(CH₃)₂

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-isopropyl)methyl-3-methoxyphenoxy]butyryl AM resinfor Fmoc-Pro Sieber ethylamide resin and omitting the coupling withFmoc-Arg(Pmc). Upon completion of the synthesis the peptide can becleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution can be concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide can be purified byHPLC using C-18 column and with a solvent mixture varying over 50minutes in a gradient from 5% to 100% acetonitrile-water containing0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Ile-Arg-NHCH(CH₃)₂ as trifluoroacetate salt.

EXAMPLE 32 N-(6-Me-nicotinyl)-Ile-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, 6-methyl-nicotinic acid for aceticacid and omitting the coupling with Fmoc-Arg(Pmc). Upon completion ofthe synthesis the peptide can be cleaved from the resin using a mixtureof (95:2.5:2.5) TFA/anisole/water for 3 hr. The peptide solution can beconcentrated in vacuo and then precipitated with diethyl ether. Thecrude peptide can be purified by HPLC using C-18 column and with asolvent mixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions can belyophilized to give N-(6-Me-nicotinyl)-Ile-Arg-NHCH₂CH₃ astrifluoroacetate salt.

EXAMPLE 33 N-(6-Me-nicotinyl)-Lys(Ac)-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, 6-methyl-nicotinic acid for aceticacid, Fmoc-Lys(Ac) for Fmoc-Ile and omitting the coupling withFmoc-Arg(Pmc). Upon completion of the synthesis the peptide can becleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution can be concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide can be purified byHPLC using C-18 column and with a solvent mixture varying over 50minutes in a gradient from 5% to 100% acetonitrile-water containing0.01% TFA. The pure fractions can be lyophilized to giveN-(6-Me-nicotinyl)-Lys(Ac)-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 34 N-Valeryl-Ile-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, valeric acid for acetic acid andomitting the coupling with Fmoc-Arg(Pmc). Upon completion of thesynthesis the peptide can be cleaved from the resin using a mixture of(95:2.5:2.5) TFA/anisole/water for 3 hr. The peptide solution can beconcentrated in vacuo and then precipitated with diethyl ether. Thecrude peptide can be purified by HPLC using C-18 column and with asolvent mixture varying over 50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01% TFA. The pure fractions can belyophilized to give N-valeryl-Ile-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 35 N-Valeryl-Lys(Ac)-Arg-Pro-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Lys(Ac) for Fmoc-Ile and valeric acid for acetic acid. Uponcompletion of the synthesis the peptide can be cleaved from the resinusing a mixture of (95:2.5:2.5) TFA/anisole/water for 3 hr. The peptidesolution can be concentrated in vacuo and then precipitated with diethylether. The crude peptide can be purified by HPLC using C-18 column andwith a solvent mixture varying over 50 minutes in a gradient from 5% to100% acetonitrile-water containing 0.01% TFA. The pure fractions can belyophilized to give N-Valeryl-Lys(Ac)-Arg-Pro-NHCH₂CH₃ astrifluoroacetate salt.

EXAMPLE 36 N-Ac-Cit-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, Fmoc-Cit for Fmoc-Ile and omitting thecoupling with Fmoc-Arg(Pmc). Upon completion of the synthesis thepeptide can be cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution can be concentrated invacuo and then precipitated with diethyl ether. The crude peptide can bepurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Cit-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 37 N-Valeryl-alloThr-Arg-NHCH₂CH₃

The procedure described in Example 1 can used but substitutingFmoc-Arg(Pbf)-[4-(4-N-ethyl)methyl-3-methoxyphenoxy]butyryl AM resin forFmoc-Pro Sieber ethylamide resin, Fmoc-alloThr(O-tBu) for Fmoc-Ile,valeric acid for acetic acid and omitting the coupling withFmoc-Arg(Pmc). Upon completion of the synthesis the peptide can becleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution can be concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide can be purified byHPLC using C-18 column and with a solvent mixture varying over 50minutes in a gradient from 5% to 100% acetonitrile-water containing0.01% TFA. The pure fractions can be lyophilized to giveN-valeryl-alloThr-Arg-NHCH₂CH₃ as trifluoroacetate salt.

EXAMPLE 38 N-Ac-Lys(Ac)-Arg-Pro-D-AlaNH₂

The procedure described in Example 1 can be used but substitutingFmoc-D-Ala-Sieber amide resin instead of Fmoc-Pro-Sieber ethylamideresin, Fmoc-Lys(Ac) for Fmoc-Ile and coupling with Fmoc-Pro beforecoupling with Fmoc-Arg(Pmc). Upon completion of the synthesis thepeptide can be cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution can be concentrated invacuo and then precipitated with diethyl ether. The crude peptide can bepurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Lys(Ac)-Arg-Pro-D-AlaNH₂ as trifluoroacetate salt.

EXAMPLE 39 N-Ac-Cit-Arg-Pro-D-AlaNH₂

The procedure described in Example 1 can be used but substitutingFmoc-D-Ala-Sieber amide resin instead of Fmoc-Pro-Sieber ethylamideresin, Fmoc-Cit for Fmoc-Ile and coupling with Fmoc-Pro before couplingwith Fmoc-Arg(Pmc). Upon completion of the synthesis the peptide can becleaved from the resin using a mixture of (95:2.5:2.5) TFA/anisole/waterfor 3 hr. The peptide solution can be concentrated in vacuo and thenprecipitated with diethyl ether. The crude peptide can be purified byHPLC using C-18 column and with a solvent mixture varying over 50minutes in a gradient from 5% to 100% acetonitrile-water containing0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-Cit-Arg-Pro-D-AlaNH₂ as trifluoroacetate salt.

EXAMPLE 40 N-Ac-alloThr-Arg-Pro-D-AlaNH₂

The procedure described in Example 1 can be used but substitutingFmoc-D-Ala-Sieber amide resin instead of Fmoc-Pro-Sieber ethylamideresin, Fmoc-alloThr(O-tBu) for Fmoc-Ile and coupling with Fmoc-Probefore coupling with Fmoc-Arg(Pmc). Upon completion of the synthesis thepeptide can be cleaved from the resin using a mixture of (95:2.5:2.5)TFA/anisole/water for 3 hr. The peptide solution can be concentrated invacuo and then precipitated with diethyl ether. The crude peptide can bepurified by HPLC using C-18 column and with a solvent mixture varyingover 50 minutes in a gradient from 5% to 100% acetonitrile-watercontaining 0.01% TFA. The pure fractions can be lyophilized to giveN-Ac-alloThr-Arg-Pro-D-AlaNH₂ as trifluoroacetate salt.

It will be evident to one skilled in the art that the present inventionis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. A compound of formula (I) (I) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅, (SEQ ID NO:1)

or a therapeutically acceptable salt thereof, wherein Xaa₁ is selectedfrom the group consisting of hydrogen and R—(CH₂)_(n)—C(O)—, wherein nis an integer from 0 to 8 and R is selected from the group consisting ofalkoxy, alkyl, amino, aryl, carboxyl, cycloalkenyl, cycloalkyl, andheterocycle; Xaa₂ is selected from the group consisting of alanyl,D-alanyl, arginyl, asparaginyl, aspartyl, citrullyl, glutaminyl,histidyl, alloisoleucyl, isoleucyl, D-isoleucyl, leucyl, D-leucyl,lysyl(N-epsilon acetyl), D-lysyl(N-epsilon acetyl),lysyl(N-epsilon-nicotinyl), N-methylisoleucyl, methionyl, norleucyl,norvalyl, ornithyl, phenylalanyl, prolyl, D-prolyl, homoseryl, seryl,allothreonyl, and threonyl; Xaa₃ is selected from the group consistingof arginyl, D-arginyl, citrullyl, histidyl, homoarginyl, lysyl,lysyl(N-epsilon isopropyl), ornithyl, and 3-(3-pyridyl)alanyl; providedthat when Xaa₃ is arginyl or D-arginyl then Xaa₂ is other than arginyl;Xaa₄ is absent or selected from the group consisting ofN-methyl-D-alanyl, 2-aminobutyryl, 2-aminoisobutyryl, D-glutaminyl,homoprolyl, hydroxyprolyl, leucyl, phenylalanyl, prolyl, and D-prolyl;and Xaa₅ is selected from the group consisting of hydroxyl,D-alanylamide, azaglycylamide, glycylamide, D-lysyl(N-epsilonacetyl)amide, —NHCH(CH₃)₂, a group represented by the formula—NH—(CH₂)_(n)—CHR¹R², and a group represented by the formula —NHR³,wherein n is an integer from 0 to 8; R¹ is selected from the groupconsisting of hydrogen, alkyl, cycloalkenyl, and cycloalkyl; R² isselected from the group consisting of hydrogen, alkoxy, alkyl, aryl,cycloalkenyl, cycloalkyl, heterocycle, and hydroxyl, with the provisothat when n is 0, R² is other than alkoxy or hydroxyl; and R³ isselected from the group consisting of hydrogen, cycloalkenyl,cycloalkyl, and hydroxyl.
 2. The compound of claim 1 wherein Xaa₁ isR—(CH₂)_(n)—C(O)—; n is 0; R is selected from the group consisting ofalkyl and heterocycle, wherein the alkyl is selected from the groupconsisting of methyl and n-butyl, and wherein the heterocycle is6-methylpyridinyl; Xaa₂ is selected from the group consisting ofD-alanyl, arginyl, asparaginyl, aspartyl, citrullyl, glutaminyl,histidyl, isoleucyl, D-isoleucyl, D-leucyl, D-lysyl(N-epsilon acetyl)lysyl(N-epsilon acetyl), lysyl(N-epsilon-nicotinyl), methionyl,norleucyl, ornithyl, prolyl, homoseryl, seryl, allothreonyl, andthreonyl; Xaa₃ is selected from the group consisting of arginyl andcitrullyl; Xaa₄ is absent or prolyl; and Xaa₅ is selected from the groupconsisting of D-alanylamide, —NHCH₂CH₃, and —NHCH(CH₃)₂.
 3. The compoundof claim 1 wherein Xaa₃ is arginyl.
 4. The compound of claim 3 whereinXaa₂ is isoleucyl.
 5. The compound of claim 4 selected from the groupconsisting of N-Ac-Ile-Arg-Pro-NHCH₂CH₃;N-(6-methylnicotinyl)-Ile-Arg-Pro-NHCH₂CH₃; N-Ac-Ile-Arg-Pro-NHCH(CH₃)₂;N-Valeryl-Ile-Arg-Pro-NHCH₂CH₃; N-Ac-Ile-Arg-Pro-D-AlaNH₂;N-Ac-Ile-Arg-NHCH₂CH₃; N-Ac-Ile-Arg-NHCH(CH₃)₂;N-(6-Me-nicotinyl)-Ile-Arg-NHCH₂CH₃; and N-Valeryl-Ile-Arg-NHCH₂CH₃. 6.The compound of claim 3 wherein Xaa₂ is D-isoleucyl.
 7. The compound ofclaim 6 selected from the group consisting ofN-(6-methylnicotinyl)-D-Ile-Arg-Pro-NHCH₂CH₃; andN-Ac-D-Ile-Arg-Pro-NHCH₂CH₃.
 8. The compound of claim 3 wherein Xaa₂ isselected from the group consisting of prolyl, D-lysyl(N-epsilon acetyl),lysyl(N-epsilon acetyl), and lysyl(N-epsilon-nicotinyl).
 9. The compoundof claim 8 selected from the group consisting ofN-Ac-Pro-Arg-Pro-NHCH₂CH₃; N-Ac-Lys(Ac)-Arg-Pro-NHCH₂CH₃;N-Ac-D-Lys(Ac)-Arg-Pro-NHCH₂CH₃; N-Ac-Lys(Ac)-Arg-NHCH₂CH₃;N-Ac-Lys(Nic)-Arg-NHCH₂CH₃; N-(6-Me-nicotinyl)-Lys(Ac)-Arg-NHCH₂CH₃;N-Valeryl-Lys(Ac)-Arg-Pro-NHCH₂CH₃; and N-Ac-Lys(Ac)-Arg-Pro-D-AlaNH₂.10. The compound of claim 3 wherein Xaa₂ is selected from the groupconsisting of D-alanyl, citrullyl, and D-leucyl.
 11. The compound ofclaim 10 selected from the group consisting ofN-Ac-D-Ala-Arg-Pro-NHCH₂CH₃; N-Ac-D-Leu-Arg-Pro-NHCH₂CH₃;N-Ac-Cit-Arg-Pro-NHCH₂CH₃; N-Ac-Cit-Arg-NHCH₂CH₃; andN-Ac-Cit-Arg-Pro-D-AlaNH₂.
 12. The compound of claim 3 wherein Xaa₂ isselected from the group consisting of arginyl, asparaginyl, methionyl,norleucyl, ornithyl, aspartyl, glutaminyl, homoseryl, histidyl, seryl,allothreonyl, and threonyl.
 13. The compound of claim 12 selected fromthe group consisting of N-Ac-Thr-Arg-Pro-NHCH₂CH₃;N-Ac-Ser-Arg-Pro-NHCH₂CH₃; N-Ac-Nle-Arg-Pro-NHCH₂CH₃;N-Ac-Orn-Arg-Pro-NHCH₂CH₃; N-Ac-Asp-Arg-Pro-NHCH₂CH₃;N-Ac-Gln-Arg-Pro-NHCH₂CH₃; N-Ac-Asn-Arg-Pro-NHCH₂CH₃;N-Ac-Met-Arg-Pro-NHCH₂CH₃; N-Ac-Hser-Arg-Pro-NHCH₂CH₃;N-Ac-alloThr-Arg-Pro-NHCH₂CH₃; N-Ac-His-Arg-Pro-NHCH₂CH₃;N-Valeryl-alloThr-Arg-NHCH₂CH₃; and N-Ac-alloThr-Arg-Pro-D-AlaNH₂. 14.The compound of claim 1 wherein Xaa₃ is citrullyl.
 15. The compound ofclaim 14 wherein Xaa₂ is selected from the group consisting of arginyland lysyl(N-epsilon acetyl).
 16. The compound of claim 15 selected fromthe group consisting of N-Ac-Arg-Cit-Pro-NHCH₂CH₃; andN-Ac-Lys(Ac)-Cit-NHCH₂CH₃.
 17. A pharmaceutical composition comprising acompound of claim 1, or a therapeutically acceptable salt thereof, incombination with a therapeutically acceptable carrier.
 18. A method ofinhibiting angiogenesis in a mammal in recognized need of such treatmentcomprising administering to the mammal a therapeutically acceptableamount of a compound of claim 1 or a therapeutically acceptable saltthereof.
 19. A method of treating cancer in a mammal in recognized needof such treatment comprising administering to the mammal atherapeutically acceptable amount of a compound of claim 1 or atherapeutically acceptable salt thereof.